1. Field of the Invention
The present invention concerns a high-speed multiple wavelength illumination source, an apparatus containing the illumination source, and methods of irradiating luminescent samples and of quantitative ratio luminescence microscopy using the illumination source and apparatus.
2. Discussion of the Background
A growing field in quantitative fluorescence microscopy is ratio imaging, which involves sequentially illuminating the same field of a specimen with two or more wavelengths of light, capturing each image, and then dividing the intensities of the two resultant images (or areas of interest within the images) to obtain an intensity ratio of the two images. In the typical case, the specimen is labeled with a fluorescent dye, the fluorescence ratio of which is related to a quantitative property of the dye or to an interaction with another substance which affects the fluorescence of the fluorescent dye. One example of a method and apparatus for multiple emission ratio photometry and multiple emission ratio imaging are described in U.S. application Ser. No. 07/935,873, filed on Aug. 26, 1992, now U.S. Pat. No. 5,332,905, the entire contents of which are incorporated herein by reference.
In quantitative fluorescence ratio imaging or fluorescence ratio photometry (also known as "quantitative ratio imaging" or "quantitative ratio photometry"), a specimen containing a fluorescent compound or species is illuminated with two different wavelengths of light (.lambda..sub.1 and .lambda..sub.2). The respective resultant fluorescent intensities (I.sub.1 and I.sub.2) are measured in one or more pre-determined areas of interest on the fluorescent specimen with a photometer. Alternatively, the fluorescent intensities I.sub.1 and I.sub.2 can be measured as an array of pixels by an imaging detector, so that an image of the fluorescent specimen can be produced and/or observed.
The relationship between the ratio of the fluorescent emission intensities (I.sub.1 /I.sub.2) at each of the two respective excitation wavelengths .lambda..sub.1 and .lambda..sub.2 is a function of the concentration of the substances which associate or interact with the fluorescent dye. The concentration of the fluorescence-affecting substance ([Substance]) is defined by the following Equation (1): ##EQU1##
The intensities may be measured at the same location(s) of the sample. Accordingly, the concentration of fluorescent compound ([Dye]) is the same at both excitation wavelengths. Thus, the only difference in measurements according to Equation (1) is the wavelength of excitation. Therefore, the dye concentration term cancels out from Equation (1), and the equation reduces to: ##EQU2##
In current practice, the rate at which the ratio image of the sample can be obtained is limited by the frequency at which the specimen can be alternately illumined with each of the excitation wavelengths. The change in wavelength is in all cases controlled by some mechanical device (such as a filter wheel) to alternate the wavelength of illumination. The most typical method of alternating the wave-length of excitation is to illuminate the sample with an arc lamp 11 (which provides a broad spectrum of light) and to change the wavelength of illumination by switching filters on a filter wheel 12 which only pass the desired wavelength of light to the specimen 13, as shown in FIG. 1.
Another method is to alternate between two sources monochromatic light focused upon the specimen. This is presently accomplished by a mechanical means 21 located between the two light sources 22 and 23 and the specimen 24, as shown in FIG. 2. Mechanical means 21 typically blocks one beam of light selectively, by either the alternate opening and closing of shutters or the rotation of a chopper wheel. After passing through mechanical means 21, the light beam passes the filter 25 or 26 before irradiating specimen 24.
The approaches employing a filter wheel, shutters or a chopper blade are limited by the speed of the mechanical movements required to change filters, shutters or chopper blades. By contrast, the speed with which a detector can be altered to make or to process a new measurement is limited only by the electronic circuitry controlling the measuring apparatus. In general, electronic impulses can be changed at a much faster rate than a mechanical device, such as a filter wheel, shutters or a chopper blade. Accordingly, it is a desirable goal in the photometric arts to provide a means for (repeatedly) changing or switching between light sources of different wavelengths at the speed of electronic circuitry, rather than at the speed of mechanical motions.
In addition, mechanically switching filters, shutters or chopper blades also causes vibrations in the measuring apparatus. Such vibrations can lead to changes in the location of the sample being measured (especially when a specimen is being viewed under a microscope), thus destroying the reliability of the data obtained from the measurements.
The fastest filter wheel presently available provides the capability to switch between different excitation wavelengths in a period of time of from about 2.5-5 milliseconds (ms). However, such a filter wheel is in constant motion during operation. The constant motion limits the duration with which the sample can be irradiated, and also introduces non-uniform interference patterns in filtering the excitation light.